A spectroscopic and mechanistic study of xanthine oxidase and xanthine dehydrogenase is proposed, focusing on the common reductive half-reaction of-these two enzymes. The subject enzymes are molybdenum hydroxylases, which are unique among monoxygenase systems in (utilizing H2O rather than 02) as the source of the oxygen atom incorporated into product and in generating (rather than consuming) reducing equivalents in the course of the reaction. Xanthine oxidase and xanthine dehydrogenase are the targets for mechanism-based inhibitors such as allopurinol, which are used to control the hyperuricemia associated with gout and gouty arthritis, as well as the genetic lesions Lesch-Nyhan syndrome and von Gierke's disease (deficiencies in hypoxanthine/guanine phosphoribityltransferase and glucose-phosphatase, respectively). The primary aim of the proposed work is to compare and contrast the chemistry whereby the molybdenum centers of xanthine oxidase and xanthine dehydrogenase catalyze the incorporation of an oxygen atom into position 8 of substrate xanthine. The work dealing with xanthine oxidase constitutes an extension of our previous studies with this enzyme, and focuses on several specific aspects of the reaction mechanism to be probed by a combination of electron paramagnetic resonance, electron spin echo and resonance Raman spectroscopies. A comprehensive solvent kinetic isotope effect study is also to be undertaken in an effort to elucidate the mechanistic role of protons in the hydroxylation reaction. Work with xanthine dehydrogenase will focus on a comparative study of the reductive half-reaction vis a vis the much better understood xanthine oxidase, including an examination of the reaction of the dehydrogenase with substrates xanthine, lumazine and 2-hydroxy-methylpurine under single- turnover conditions. The pH dependence of the behavior of xanthine dehydrogenase in the course of reductive titrations, and the spectroscopic characteristics of oxidized enzyme will also be investigated. A comparative study of the flavin centers of the oxidase and dehydrogenase utilizing resonance Raman spectroscopy will also be undertaken.